JP2002523334A - In vivo cell cycle alterations specifically triggering tumor cell apoptosis - Google Patents

Abstract

  (57) [Summary] First substances (eg, peptide aldehydes LLnL and LVP) inhibit the function of the proteasome in cells. A second substance (e.g., rampirnase) inhibits protein synthesis in the cell or increases the intracellular expression of a cyclin-dependent kinase (CDK) inhibitor. Both substances are administered in such a way that their actions are simultaneous. Thereby, the cell cycle of the modified tumor cells is stopped, and the cells die by apoptosis.

Description

DETAILED DESCRIPTION OF THE INVENTION

[0001]

BACKGROUND OF THE INVENTION

The present invention relates to cell biology, and more particularly to apoptosis. Most directly, the present invention relates to a method of inducing apoptosis in tumor cells in vivo. It has long been known that cancer tumors arise from the dysregulated growth of undifferentiated tumor cells. To that end, scientists have long sought treatments that would not allow tumor cells to grow.

To achieve this, scientists have developed N-acetyl-leucinyl-leucinyl-, a peptide aldehyde known to inhibit the function of the proteasome.
Attempts have been made to disrupt the cell cycle of proliferating HL-60 tumor cells by treatment with norleucinal (LLnL). This stops HL-60 cells in the G1 phase of the cell cycle and prevents them from entering the S phase. That is, if such cells do not advance to the next stage of the cell cycle, they die (this is a phenomenon known as programmed cell death or apoptosis).

[0003] However, such studies, namely the function of the proteasome (which
It has been narrowly concentrated only on aspects of the cell cycle that inhibit it (provided to be necessary for normal cell cycle progression). In fact, cell progression along the cell cycle occurs as a result of the most complex equilibrium mechanisms. At any point in the cell cycle, a cell is a biological mechanism that tends to progress the cell along the cell cycle as well as a biological mechanism that tends to hold the cell in any phase of the cell cycle in which it is currently. Attached to the mechanism. The cells are in fact, eg, from G1 phase to S phase or from S phase to G2 phase.
Whether or not to progress to mitosis (M phase) from G2 phase or from G2 phase
It is determined by the balance of the biological forces acting on the cells. The positive effects of, for example, cyclins, which act to stimulate progression in the cell cycle, predominate over the negative effects of, for example, kinase inhibitors, which act to stop cells during the existing cell cycle If so, the cell enters or leaves S phase. Otherwise, the cells will not progress this way.

The present invention began with the recognition that existing approaches to promoting tumor cell apoptosis do not utilize such positive and negative effects simultaneously. As a result, such an approach is not as effective, even if it is another.

According to the present invention, therapeutically effective doses of two substances are administered in vivo in such a way that their effects are simultaneous. The first substance stops the cell at a particular phase of the cell cycle and the second substance prevents the cell from proceeding beyond this phase. Thus, the equilibrium mechanism described above is used to most effectively block tumor cell growth and thereby induce apoptosis.

[0006] According to a preferred embodiment, the first substance inhibits proteasome function in a cell or inhibits activation of NFκB or both. If the function of the proteasome in the cell is inhibited, the activation of NFκB in the cell is similarly inhibited. This is because NFκB is a heterodimer with a protein known as p50 as one of its components. Normally, the p50 protein is produced by proteolytic processing of its precursor protein p105. This processing is a function of the proteasome. When proteasome function is inhibited, no p50 protein is produced, and active N5 from other proteins known as p50 and p65 proteins.
Instead of producing FκB heterodimers, inactive heterodimers are produced from p105 and p65 proteins. In addition, if proteasome function is inhibited, this prevents proteolytic degradation of the IκB protein. This IκB protein inhibits NFκB function by complexing with NFκB and preventing NFκB from entering the nucleus of the cell. In other words, inhibition of proteasome function also inhibits NFKB function by reducing the extent to which inhibitors of NFKB are destroyed. Two peptide aldehydes, namely
LLnL and N-acetyl-leucinyl-valinyl-phenylalaninal (
LVP) is suitable for this purpose.

[0007] The stage at which inhibition of proteasome function in a cell arrests the cell cycle can vary.
As described above, in HL-60 cells, arrest occurs in G1 phase, but DU-14
In 5 human prostate carcinoma cells, arrest occurs in G2 phase. According to a preferred embodiment, the second agent inhibits intracellular protein synthesis or increases the intracellular expression of a cyclin-dependent kinase (CDK) inhibitor such as p27KIP1. Ranpirnase is suitable for this purpose. (Rampirnase is the USAN-approved generic name for the drug described in U.S. Patent No. 5,559,212, now known as ONCONASE.)

[0008] As long as the substances are administered in such a way that the actions of these substances are effected simultaneously, the simultaneous action described above means that both substances are administered separately (by injection, orally or both). Can be achieved by doing The above described simultaneous effects can also be achieved by administering both substances simultaneously (as in a mixture or other form containing both substances).

[0009]Detailed description of preferred embodiments  In vitroThe experiments were performed using five drugs on three cell lines. medicine
Agents include ranpirnase, doxorubicin, LLnL, LVP and N-acetyl
-Leucinyl-leucinyl-methioninal (LLM) and the cell line is DU
-145 human prostate carcinoma, A-549 human lung carcinoma and MDA-MB-231 human
It is breast carcinoma.In vivoThe experiment was performed in nude mice using ranpirnase,
Using Xorubicin and LLnL and MDA-MB-231 human breast carcinoma cell lines
I went for it.

In vitro Materials and Methods: Cell Lines The DU-145, A-549 and MDA-MB-231 cell lines were obtained from American T.
Purchased from ype Culture Collection. A-549 cells were obtained from Cell Tissue Kinet
23: 237, 1990. The DU-145 cell line contains 1
0% fetal bovine serum (FBS), 1% glutamine and 1% Pen-Strep-Fungizone
Eagle supplemented with minimal essential medium. The number of DU-145 cells used in the MTT assay was 10,000 cells / well. MDA
-The MB-231 cell line contains 10% FBS, 1% glutamine and 1% Pen-Strep-
Maintained in Leibovitz's L-15 medium supplemented with Fungizone. The number of MDA-MB-231 cells used in the MTT assay was 10,000 cells /
Well. All agents were obtained from JRH Bioscience, Lenexa, KS. Cell number determination, description of the MTT colorimetric assay and statistical analysis (including evaluation of synergistic interactions) are described in Cell Tissue Kinet 23: 237, 1990 and Adv Cancer Res 35: 269,
1981.

In vitro drug Rampirnase was dissolved as described in Cell Tissue Kinet 23: 237, 1990. LLnL, LLM and N-acetyl-leucinyl-valinyl-
Phenylalaninal (LVP) was purchased from Sigma Corporation, St Louis, MO. All three materials were dissolved in 100% ethanol to make a 1 mM stock solution. This stock solution was diluted in the growth medium of the cell line being tested to produce a solution that was added to the plate containing cells for the MTT assay.

In vitro experimental results Rampirnase and LLnL were obtained from DU-145, A-549 and MDA-
Interacts synergistically with the MB-231 cell line (Tables 1 and 2). A-5
In 49 and DU-145 cell lines, the antitumor activity of ranpirnase was also increased by LVP, but not by LLM (Table 3).
The activity of ranpirnase on the MDA-MB-231 cell line was 25 μM LLM.
The did not (mean ED ₅₀ with only be influenced to a minimum by an additional treatment with,
(Reduced to about 2-fold) (Table 3). These findings indicate that Proc Natl Acad Sci USA 94: LLM peptide does not significantly inhibit proteasome activity.
855-860, consistent with the report published in 1997.

[0013] In MDA-MB-231 cells, ranpirnase is present at 0.1 to 10.0 μm.
ranpirnase in the range of g / ml and LLn in the range of 5-25 μM
It interacts synergistically with LLnL at final concentrations of L, especially at LLnL concentrations in the range between 10-25 μM. The average ED ₅₀ value for rampirnase ranges from 1.434 μg / ml (rampirnase alone) to 1 for each of LLnL.
Reduction to 0.037, 0.022 and 0.001 μg / ml for rampirnase in combination with LLnL at 0, 15 and 25 μM concentrations (Table 2) In addition, 0 μl with or without 15 μM LLnL
A combination of .1 μg / ml (about 0.17 μM) of doxorubicin and ranpirnase was tested (Table 4). The mean ED ₅₀ value of ranpirnase when used in combination with doxorubicin was reduced 2.2-fold compared to the mean ED ₅₀ value of ranpirnase alone. Under these conditions, ranpirnase + doxorubicin +
Rampirnase + LL without doxorubicin using triple combination of LLnL
Addition of doxorubicin does not significantly reduce cell proliferation or cell viability when compared to nL (Table 4).

[0014] In A-549 cells, the combination of rampirnase and LLnL is:
It was synergistic at high concentrations of LLnL (15-35 μM) (Table 1). The combination of low concentration (0.1-1.0 μg / ml) of rampirnase and low concentration (10 μM) of LLnL was not synergistic, but the effect of this combination was greater than that of rampirnase alone (Table 1). 2). The average ED ₅₀ value for rampirnase is 3.94.
From 4 μg / ml (rampirnase alone), 10, 15, 25 and 35 respectively
1. For the combination of LLnL and rampirnase at a LLnL concentration of μM.
356, 0.640, 0.060 and 0.001 μg / ml or less (Table 2
).

In DU-145 cells, ranpirnase interacted synergistically with LLnL over all concentrations of ranpirnase and 10-25 μM concentration of LLnL (Table 1). Rampirnase alone and 10, 15, and 25 μM L
The ED ₅₀ value for each of the ranpirnase combined with LnL is 42.276.
, 1.231, 0.001 and <0.001 μg / ml (Table 2). To test whether the observed antitumor activity of the combination was due to apoptosis, an excess (50 μM final concentration) of Ac-DEVD-cho (N-acetyl-
L-aspartyl-L-glutamyl-L-valinyl-L-aspartal) was added to overcome the perceived difficulty in its ability to penetrate plasma membranes (Ac-DEVD-ch).
o was chosen because it is a known inhibitor of caspases.
Caspases are cysteine-proteases involved in apoptotic cell death). Ac-DEVD-cho increased the viability of cells treated with the combination of rampirnase and 25 μM LLnL about 2-3 fold. Under the same conditions without rampirnase, the combination of LLnL and Ac-DEVD-cho increased cell viability about 4-fold compared to cells treated with LLnL alone (Table 5). This suggests that at least part of the observed antitumor activity is the development of apoptotic cell death.

To establish whether the observed differences between the antitumor activity of ranpirnase as a single agent and ranpirnase combined with the respective peptide inhibitors were significant, Newman-Cruise statistical Performed analysis (Table 6)
. As shown in the table, in all tumor cell lines tested, the antitumor activity of ranpirnase in combination with the LLnL proteasome inhibitor was significantly greater than the antitumor activity of each agent used as a single agent. Met. 25 μl on proliferation and viability of MDA-MB-231 cells when compared to untreated controls
There was no significant effect of the MLLM peptide, and there was no significant difference between the activity of the combination of ranpirnase and 25 μM LLM peptide and the activity of ranpirnase alone (Table 6). In flow cytometry studies of DU-145 cells, apoptosis appears to coincide with the G2 arrest of the cell cycle.

In Vivo Experimental Results In vivo experiments (see Table 7) were performed on nude mice bearing MDA-MB-231 tumors to combine the activity of ranpirnase and LLnL as a single agent with the activity of combined ranpirnase and LLnL. And compared. The doses of rampirnase and LLnL were 2.5 mg / kg and 5 mg / kg of mouse body weight, respectively.

In the experiments described above, ranpirnase and LLnL were administered individually in vivo intraperitoneally by a continuous injection twice a week for three weeks on a twice weekly schedule. Other dosage forms with simultaneous effects (eg, with or without other agents that enhance delivery to the intended site in the body) can also be used. Furthermore, it is now possible to administer the substances of the invention in a single dosage form, with or without the addition of one or more other agents that enhance delivery to the intended site in the body. Although expected to be most advantageous, this has not yet been experimentally proven.

While at least one preferred embodiment of the present invention has been described above, this is not limiting and is merely illustrative. The scope of the present invention is limited only by the claims.

[0020]

[Table 1]

[0021]

[Table 2]

[0022]

[Table 3]

[0023]

[Table 4]

[0024]

[Table 5]

[0025]

[Table 6]

[0026]

[Table 7]

[0027]

[Table 8]

[0028]

[Table 9]

Claims (30)

[Claims] 1. The method according to claim 1, wherein the cells are arrested at a predetermined phase of the cell cycle by administering a therapeutically active dose of the first substance, and at the same time administering a therapeutically active dose of the second substance. Preventing the cells from progressing beyond this predetermined phase of the cell cycle by altering the cell cycle of the cells in a living patient. 2. The cell is arrested at a given phase of the cell cycle by administering a therapeutically active dose of a first substance, and at the same time administering a therapeutically active dose of a second substance. Preventing the cells from progressing beyond this predetermined phase of the cell cycle by inducing tumor cell apoptosis in vivo. 3. A method for administering to a patient a therapeutically active dose of a first substance that inhibits intracellular proteasome function, and a method of treating a second substance that acts to inhibit intracellular protein synthesis. Administering to the patient a dose that is more active, and performing these administration steps in such a way that the effects are simultaneous, to alter the cell cycle of cells in a living patient. 4. The method according to claim 3, wherein the first substance is a peptide. 5. The method according to claim 4, wherein the first substance is a peptide aldehyde. 6. The method according to claim 5, wherein the peptide aldehyde is N-acetyl-leucinyl-leucinyl-norleucinal (LLnL). 7. The method according to claim 5, wherein the peptide aldehyde is N-acetyl-leucinyl-valinyl-phenylalaninal (LVP). 8. The method according to claim 3, wherein the second substance is a ribonuclease (RNase). 9. The method according to claim 8, wherein the RNase is ranpirnase. 10. A method for administering to a patient a therapeutically active dose of a first substance that inhibits intracellular activation of NFκB, and treating a second substance that acts to inhibit intracellular protein synthesis. A method of altering the cell cycle of cells in a living patient, comprising administering to the patient a therapeutically active dose, and performing these administration steps in such a manner that their effects are simultaneous. 11. The method according to claim 10, wherein the first substance is a peptide. 12. The method according to claim 11, wherein the first substance is a peptide aldehyde. 13. The method according to claim 12, wherein the peptide aldehyde is N-acetyl-leucinyl-leucinyl-norleucinal (LLnL). 14. The method according to claim 12, wherein the peptide aldehyde is N-acetyl-leucinyl-valinyl-phenylalaninal (LVP). 15. The method according to claim 10, wherein the second substance is a ribonuclease (RNase). 16. The method according to claim 15, wherein the RNase is rampirnase. 17. A method for administering to a patient a therapeutically active dose of a first substance that inhibits intracellular proteasome function and increases the intracellular expression of a cyclin-dependent kinase (CDK) inhibitor. Administering a therapeutically active dose of a substance to a patient, and performing these administration steps in such a way that their effects are simultaneous, altering the cell cycle of cells in a living patient Method. 18. The method according to claim 17, wherein the CDK inhibitor is p27KIP1. 19. The method according to claim 18, wherein the first substance is a peptide. 20. The method according to claim 18, wherein the first substance is a peptide aldehyde. 21. The method according to claim 20, wherein the peptide aldehyde is N-acetyl-leucinyl-leucinyl-norleucinal (LLnL). 22. The method according to claim 20, wherein the peptide aldehyde is N-acetyl-leucinyl-valinyl-phenylalaninal (LVP). 23. The method according to claim 18, wherein the second substance is a ribonuclease (RNase). 24. The method according to claim 23, wherein the RNase is ranpirnase. 25. The cell is arrested in the G2 phase of the cell cycle by inhibiting intracellular proteasome function, and at the same time, increasing the intracellular expression of a cyclin-dependent kinase inhibitor to cause the cell to enter the cell cycle. A method for inducing apoptosis of in vivo tumor cells, comprising the step of preventing progression beyond the G2 phase. 26. A method for administering to a living patient a therapeutically active dose of a peptide that inhibits intracellular proteasome function and a therapeutically active dose of a different substance that inhibits intracellular protein synthesis. A method of inducing apoptosis of an in vivo tumor cell, wherein said steps of administering are performed in such a manner that their effects are simultaneous. 27. A different substance that administers a therapeutically active dose of a peptide that inhibits intracellular proteasome function to a living patient and increases the intracellular expression of a cyclin-dependent kinase (CDK) inhibitor Administering to a patient a therapeutically active dose of and administering these administration steps in such a way that their effects are simultaneous. 28. The peptide is N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) or N-acetyl-leucinyl-valinyl-phenylalaninal (LVP), and the different substance is ribonuclease (RNase). A method according to claim 26 or 27. 29. A therapeutically active dose of a peptide selected from the group consisting of N-acetyl-leucinyl-leucinyl-norleucinal (LLnL) and N-acetyl-leucinyl-valinyl-phenylalaninal (LVP). In vivo tumor cell apoptosis comprising administering to a living patient, and administering to the patient a therapeutically active dose of rampirnase, and performing these administration steps in such a way that their effects are simultaneous. How to trigger. 30. The method of claim 29, wherein the administering step is performed by intravenously injecting rampirnase and the selected peptide.